1. Field of the Invention
The present invention is a process to transform medroxyprogesterone acetate (I) to 11xcex2,17xcex1,21-trihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 21-acetate (VI).
2. Description of the Related Art
The microbial enzymatic xcex941-dehydrogenation of steroids is known to those skilled in the art. Biotechnology and Bioengineering, 37, 97-102 (1991) discloses the xcex941-dehydrogenation of 6xcex1-methylhydrocortisone 21-acetate by Arthrobacter simplex in an organic solvent.
U.S. Pat. No. 4,684,610 discloses a process for converting 1,2-saturated steroids to 1,2-dehydro, steroids by contacting the 1,2-saturated steroid with A. simplex or Bacillis cyclooxydans in the presence of exogenous electron carrier and a water-immiscible aromatic hydrocarbon solvent.
U.S. Pat. No. 4,749,649 discloses the use of scavengers of toxic oxygen species in the microbial xcex941-dehydrogenation of steroids.
The microbial enzymatic 11xcex2-hydroxylation of steroids with microorganisms such as Curvularia lunata is known to those skilled in the art, see for example U.S. Pat. No. 3,419,470. The concentration of the substrate undergoing 11-xcex2 hydroxylation is quite low. For example, U.S. Pat. No. 3,530,038 discloses a maximum concentration of steroid substrates 17xcex1-acetoxypregn-4-en-21-ol-3,20-dione and 17xcex1,21-diacetoxypregn-4-ene-3,20-dione is 0.5 g/L. European Patent EP 0 042 451 A1 again discloses that the amount of steroid substrate concentration does not exceed 0.5 g/L. U.S. Pat. No. 4,353,985 does not give specific examples, and uses 17xcex1,21-orthoesters. U.S. Pat. No. 4,588,683 discloses that the steroid substrate concentration of 17xcex1,20xcex2,21-trihydroxypregn-4-en-3-one is 0.5 g/L. U.S. Pat. No. 4,898,693 discloses that the steroid substrate concentration of 6xcex1-fluoro-17xcex1-hydroxy-16xcex1-methylpregn-4-en-3,20-dione is 0.4 g/L. All but the last patent listed above relate to eliminating unwanted 14xcex1-hydroxylation resulting in high yields of 11xcex2-hydroxylated product (80-92%). However, the substrate charge to the fermentation does not exceed 0.5 g/L. Japanese Published Application 62-118898 discloses the use of C. lunata MCI1690 (registered at Microbial Industry Institute strain No. 8515) at a substrate concentration of 10 g/L but with substance S and analogs thereof, not the substrate of the present invention. The process of the present invention uses a different culture than that of the Japanese 62-118898 and provides high yields using high substrate concentrations.
The functionalization of the C21-methyl group of pregnanes followed by displacement with acetate to produce the corresponding 21-acetate is known to those skilled in the art. GB 2,318,790 discloses the transformation of the C21-methyl group of a xcex941-11xcex2-hydroxy steroid to the corresponding 21-hydroxy steroid by functionalization with one bromine atom followed by displacement with acetate. The process of the present invention does not use bromine.
GB 2,318,790 discloses the transformation of 17xcex1-hydroxy-6xcex1-methylpregn-4-ene-3,20-dione 17-acetate (I) to 11xcex2,17xcex1-dihydroxy-21-diiodo-6xcex1-methylpregna-1,4-diene-3,20-dione (V) by microbial xcex941-dehydrogenation by use of Nocardia simplex, microbial 11xcex2-hydroxylation by use of C. lunata and 21-hydroxylation by use of bromine. The present invention transforms 17xcex1-hydroxy-6xcex1-methylpregn-4-ene-3,20-dione 17-acetate (I) to 11xcex2,17xcex1-dihydroxy-21-diiodo-6xcex1-methylpregna-1,4-diene-3,20-dione (V) but does not use bromine.
Disclosed is a process for the preparation of 11xcex2,17xcex1,21-trihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 21-acetate (VI) which comprises:
(1) contacting 17xcex1-hydroxy-6xcex1-methylpregn-4-ene-3,20-dione 17-acetate (I) with a xcex941-dehydrogenase to produce 17xcex1-hydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17 acetate (II);
(2) contacting 17xcex1-hydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17 acetate (II) with a 11xcex2-hydroxylase to produce 11xcex2,17xcex1-dihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17-acetate (III);
(3) hydrolyzing the 11xcex2,17xcex1-dihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17-acetate (III) to producel 11xcex2,17xcex1-dihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione (IV);
(4) contacting 11xcex2,17xcex1-dihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione (IV) with iodine, a catalyst, a mild base to produce 11xcex2,17xcex1-dihydroxy-21-diiodo-6xcex1-methylpregna-1,4-diene-3,20-dione (V) and
(5) contacting 11xcex2,17xcex1-dihydroxy-21-diiodo-6xcex1-methylpregna-1,4-diene-3,20-dione (V) with a salt of acetic acid.
Also disclosed is a diiodo steroid of the formula: 
Further disclosed is a process for the removal of residual unhydroxylated material which comprises:
(1) contacting 17xcex1-hydroxy-6xcex1-methylpregn-4-ene-3,20-dione 17-acetate (I) with a xcex941-dehydrogenase to produce 17xcex1-hydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17 acetate (II),
(2) crystallizing the 17xcex1-hydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17 acetate (II) produced from a nonpolar solvent or solvent mixture.
Additionally disclosed is a process for the purification of 11xcex2,17xcex1-dihydroxy-6xcex1-methylpregna-1,4-diene-3,20-dione 17-acetate (III) produced by a 11xcex2-hydroxylase which comprises:
(1) crystallization from a solvent selected from the group consisting of toluene, benzene, xylene, n-butyl acetate and mixtures thereof with hydrocarbon solvents selected from the group consisting of hexane, heptane, isooctane, cyclohexane and methylcyclohexane.